Staphylococcus aureus is both a skin commensal and also a formidable pathogen, causing most skin and soft tissue and surgical site infections in the United States. Skin infections due to S. aureus also serve as a site from which disease can disseminate to virtually any organ, making this pathogen one of the primary culprits underlying severe sepsis, necrotizing pneumonia and bone, joint, and heart valve infections. In addition, antibiotic-resistant strains such as Methicillin Resistant Staphylococcus aureus (MRSA) are more difficult and costly to treat. A particularly important gap in our knowledge is understanding how S. aureus colonizes and survives on the epithelial cells of the skin, and how it breaches epithelial barriers during infection. Understanding these processes may allow us to decrease colonization and disease burden. We hypothesize that S. aureus virulence factors have specific functions in the skin during colonization and in other epithelia during infection. All invasive strains of S. aureus carry the gene for ?-toxin, and MRSA strains produce increased amounts of toxin. Antibody titers to ?-toxin are protective from invasive disease, and experimental infection with ?-toxin-deficient mutants have reduced virulence in models of skin infection and pneumonia. We performed a genetic screen for novel host factors that control susceptibility to ?-toxin by intoxicating mutagenized haploid human cells and selecting for mutants that are resistant to cell death caused by the toxin. Our screen identified a previously unrecognized set of genes of the epithelial cell-cell adherens junctions that modulate ?-toxin susceptibility. The adherens junctions are important for epithelial barrier function, tissue integrity, and wound healing. We hypothesize that altering expression of some of these genes will reduce ?- toxin virulence in the skin and reduce morbidity from MRSA infection. In this proposal we will use human keratinocytes, polarized MDCK epithelial cells, and a transgenic mouse line to study how the adherens junctions control MRSA pathogenicity. For in vitro modeling of S. aureus skin infections, we developed a 3D organotypic culture model of human skin that can be colonized and infected with MRSA. Results from the experiments proposed here will be the basis of a novel area of potential host targets at the epithelial junctions that modulate MRSA colonization and morbidity.